Over recent years, it has become apparent that mouse antibodies are not ideal reagents for in vivo use due to induction of human anti-mouse responses in recipient patients. A number of solutions have been proposed including the production of chimeric and humanized antibodies (Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and Winter, U.S. Pat. No. 5,225,539).
Human monoclonals antibodies are advantageous compared with those from mouse or other species, because, inter alia, they exhibit little or no immunogenicity in a human host. However, conventional technology for producing murine monoclonals cannot be applied unmodified to production of human antibodies for several reasons. First, mouse procedures typically involve sacrificing the mouse, a procedure that is obviously unacceptable to humans. Second, humans cannot be immunized with many types of antigens, including human antigens, due to the risk of inducing an undesired immune response. Third, forming immortalized derivatives of human B cells has proved more difficult than for mouse B cells.
Early techniques for producing human antibodies met with only limited success. For example, immortalization of immunized human lymphocytes with Epstein-Barr virus, while successful in forming monoclonal-antibody secreting cultures, has often failed to produce cells having sufficiently long lifespans to provide a reliable source of the desired antibody. Kozbor et al. (1982), Hybridoma 1:323. In another approach, hybridomas generated by fusion of immunized human lymphoid cell lines with mouse myelomas, were found to exhibit chromosomal instability. Nowinski et al. (1980), Science 210:537; Lane et al. (1982), J. Exp. Med. 155:133 (1982).
Another approach has been described by Ostberg et al. (1983), Hybridoma 2:361-367 and Engelman et al., U.S. Pat. No. 4,634,666. This method entails fusing a mouse myeloma cell with a nonimmunized human B-lymphocyte to form a xenogenic fusion cell. The fusion cell is then fused with an immunized human B-lymphocyte to produce a trioma cell. A number of human monoclonal antibodies to viral pathogens have been isolated using this approach.
A further approach has used the phage display technique to screen libraries of immunoglobulin genes obtained directly from human lymphatic cells from a naive human. A basic concept of phage display methods is the establishment of a physical association between DNA encoding an antibody to be screened and the antibody chain. This physical association is provided by the phage particle, which displays an antibody as part of a capsid enclosing the phage genome which encodes the antibody. The establishment of a physical association between antibodies and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different antibodies. Phage displaying an antibody with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of antibodies displayed from these phage can be determined from their respective genomes. Using these methods an antibody identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. Although the phage display method provides a powerful means of selection, the number of potential antibodies to be analyzed in a naive human library is very large, about 1012. Further, many of the antibodies in such a library are nonnaturally occurring combinations of heavy and light chain resulting from the random manner in which populations of these chains are combined when being cloned into the phage display vector. Such nonnaturally occurring combinations often lack capacity for strong binding. Thus, desired human antibodies with strong affinity for a human antibody are typically rare and consequently difficult to isolate from such libraries.
Human antibodies can also be produced from non-human transgenic mammals having transgenes encoding human immunoglobulin genes and having an inactivated endogenous immunoglobulin locus. The transgenic mammals resulting from this process are capable of functionally rearranging the immunoglobulin component sequences, and expressing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes, without expressing endogenous immunoglobulin genes. The production and properties of mammals having these properties are reported by, e.g., Lonberg et al., WO93/12227 (1993); U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) (each of which is incorporated by reference in its entirety for all purposes). Antibodies are obtained by immunizing a transgenic nonhuman mammal, such as described by Lonberg or Kucherlapati, supra, with antigen Monoclonal antibodies are prepared by fusing B-cells from such mammals to suitable myeloma cell lines using conventional Kohler-Milstein technology.
The present application is related to PCT 98/06704, filed, Apr. 3, 1998, U.S. Ser. No. 08/835,159, filed Apr. 4, 1997 and U.S. Ser. No. 08/832,985, filed Apr. 4, 1997, all of which are incorporated by reference in their entirety for all purposes.
The invention provides methods of producing a human antibody display library. Such methods entail providing a nonhuman transgenic animal whose genome comprises a plurality of human immunoglobulin genes that can be expressed to produce a plurality of human antibodies. A population of nucleic acids encoding human antibody chains is isolated from lymphatic cells of the nonhuman transgenic animal. The nucleic acids are then introduced into a display vector to provide library of display packages, in which a library member comprises a nucleic acid encoding an antibody chain, and the antibody chain is displayed from the package.
The invention also provides methods of producing a human Fab phage display library. Such methods entail providing a nonhuman transgenic animal whose genome comprises a plurality of human immunoglobulin genes that can be expressed to produced a plurality of human antibodies. Populations of nucleic acids respectively encoding human antibody heavy chains and human antibody light chains are isolated from lymphatic cells of the nonhuman transgenic animal. The populations are cloned into multiple copies of a phage display vector to produce a display library, in which a library member comprises a phage capable of displaying from its outersurface a fusion protein comprising a phage coat protein, a human antibody light chain or human antibody heavy chain. In at least some members, the human antibody heavy or light chain is complexed with a partner human antibody heavy or light chain, the complex forming a Fab fragment to be screened.
The invention further provides libraries of at least ten different nucleic segments encoding human antibody chains. At least 50% of segments in the library encode human antibody chains showing at least 108 Mxe2x88x921 affinity for the same target and no library member constitutes more than 50% of the library. In some libraries, at least 90% of the pairs of different nucleic acid segments encode heavy and light chains that form complexes having at least 109 Mxe2x88x921 affinity of the target.
The invention further provides libraries of at least ten different nucleic segments encoding human antibody chains, in which at least 90% of segments in the library encode human antibody chains for the same target and no library member constitutes more than 50% of the library, and the library is free of segments encoding human lambda light chains.
The invention also provides libraries of at least 1000 different nucleic segments encoding human antibody chains, in which at least 90% of segments in the library encode human antibody chains for the same target and no library member constitutes more than 50% of the library, wherein each segment comprises subsequence(s) from a human VH and/or a human VL gene, and no more than 40 human VH genes and no more than 40 human VL genes are represented in the library.
The invention further provides libraries of at least ten types of human antibodies in which at least 50% of the types of human antibodies in the library have an affinity of at least 1010 Mxe2x88x921 for the same target and no type of library member constitutes more than 25% of the library.